Fluorescence (FL) techniques have emerged as a mainstream research and development area in science and engineering, particularly in the field of biochemical and biological science. Currently, fluorescent molecules are used as probes for DNA sequencing, fluorescence-activated cell sorting, high-throughput screening, and clinical diagnostics.
Fluorescence-based techniques offer high sensitivity, low background noise and broad dynamic ranges. A great number of fluorescent probes have been investigated and are already widely used in biotechnology. Many of them show favorable spectral properties of visible absorption and emission wavelength, high extinction coefficients, and reasonable quantum yields. Upon complexation with proteins and DNA, the fluorescence of the bioprobes can be enhanced/quenched and/or red/blue-shifted, thus enabling visual observation of the biomacromolecular species. Among these, the most useful probes are those that act as “turn-on” sensors, whose fluorescence is activated by the analytes.
Several probes for DNA detection based on fluorescent enhancement have been developed such as phenanthridine and acridine derivatives. Middendorf et al. have reported on ethidium bromide (EB), a well-known phenanthridine derivative, which has already been widely used for DNA-sequencing (U.S. Pat. No. 4,729,947, U.S. Pat. No. 5,346,603, U.S. Pat. No. 6,143,151, U.S. Pat. No. 6,143,153). FL enhancement induced by proteins can be attributed to the interaction with hydrophobic regions of proteins, such as NanoOrange (Molecular Probes, Inc., U.S. Pat. No. 6,818,642) and Nile red (U.S. Pat. No. 6,897,297, U.S. Pat. No. 6,465,208), or reaction with amine groups of proteins in the presence of cyanide or thiols, such as fluorescamine (U.S. Pat. No. 4,203,967) and o-phthaldialdehyde (U.S. Pat. No. 6,969,615, U.S. Pat. No. 6,607,918). The FL of cyanine dyes has been found to increase dramatically upon complexation with DNA and proteins. (U.S. Pat. No. 5,627,027, U.S. Pat. No. 5,410,030). Haugland et al. have reported unsymmetrical cyanine dyes, which possess superior fluorescent characteristics when complexed with nucleic acids (U.S. Pat. No. 5,436,134). The SYPRO® dyes are merocyanine dyes that are essentially non-fluorescent when free in solution but become intensely fluorescent in hydrophobic environments (e.g. SYPRO®Red and SYPRO®Orange dyes of Molecular Probes, Inc., U.S. Pat. No. 6,914,250, U.S. Pat. No. 6,316,267). Water-soluble cyanine dyes, such as Cy3 and Cy5, are commonly used in labeling of DNA or RNA for microarray (V. R. Iyer et al., Science, 1999, 283, 83). Cy3 and Cy5 have merits of high fluorescence intensity and emission even in the solid state; however, they are quite unstable and show insufficient detection sensitivity (U.S. Pat. No. 7,015,002).
As described in U.S. Pat. No. 7,109,314, a good fluorescent dye should possess a high fluorescent quantum yield and molecular absorption coefficient, as well as good solubility in aqueous media and stability under ambient conditions. However, most of the dyes discussed above are lipophilic, which are at best, only dispersible in aqueous media. For example, Nile Red, a dye used to stain proteins, should be first dissolved in acetone and then mixed rapidly with water immediately prior to use (J. R. Daban et al, Anal. Biochem, 1991, 199, 169).
Additionally, substantially all of the above-described fluorescent dyes suffer from the problem of aggregation-caused quenching (ACQ). Due to their lipophilic character, these fluorescent dyes are prone to aggregate when dispersed in aqueous media or when bound to biological macromolecules. The close proximity of the chromophores often induces a non-radiative energy transfer mechanism that results in self-quenching of the luminescence. This self-quenching drastically reduces the dyes' fluorescent signal thereby prohibiting their use as efficient bioprobes or biosensors.
Substantial effort has been made to mitigate aggregate formation of these dyes (J. R. Lakowicz, et al. Anal Biochem, 2003, 320, 13). However, only a small number of researchers have focused on the design and synthesis of novel organic molecules or polymers that do not suffer from fluorescent quenching, and moreover, even display enhanced light emission upon aggregation.
Recently, aggregation-induced emission (AIE) has been observed. This phenomena is exactly opposite of ACQ. Some non-emissive dyes can be induced to emit efficiently by aggregate formation. AIE molecules with high quantum yields ΦF (up to 0.85) and various emission colors (blue, green, yellow and red) have been reported. While the AIE dyes have been used for the construction of efficient optical and photonic devices, the possibility of employing them as bioprobes for detecting biopolymers has been virtually unexplored. Accordingly, there remains a great need for water-soluble “light-up” compounds and probes, for example, for the detection of biomacromolecules such as DNA and proteins.
Many known fluorescent materials accomplish the detection of saccharides by the competing intramolecular interaction of an amine functionality with a boronic acid pendant. Less effort has been spent on the detection of other biological compounds. Furthermore, vapor-sensing compounds and devices are often manufactured from expensive platinum salts and complexes and/or in combination with palladium. They are based mainly on a color shift from dark-red to light-red, making it difficult to visually sense the color shift. Sensors exhibiting an on-off change in their luminescent color rather than a color shift will be thus not only advantageous but also more sensitive. To applicants' knowledge, the only known “on-off” example was shown by Kato (U.S. Pat. No. 6,822,096), who utilized the luminescence change from the invisible near-infrared to the visible red of binuclear platinum (II) complexes. However, these complexes only shift the emitted wavelength out of the visible spectrum.
Fluorescent materials, including inorganic semiconductor quantum dots, organic and metallorganic dyes, dye-doped silica or polymer particles, have currently attracted great attention in a wide variety of high-technology applications such as high-throughput screening, ultra-sensitive assays, optoelectronics, and living cell imaging. Colloidal quantum dots (hundreds to thousands of atoms) are traditionally made from crystals of IIA-VIA or IIIB-VB elements (PbS, CdSe, etc.) or other semiconductors. The heavy metals therein are intrinsically toxic to the researchers and the experimental systems (e.g., living cells), as well as generating a toxic waste stream into the environment. Organic and metallorganic dyes generally consist of π-conjugated ring structures such as xanthenes, pyrenes or cyanines, with emissions across the spectrum from UV to the near infrared (˜300-900 nm) and may be fine tuned to particular wavelengths or applications by changing the chemistry of their substituent groups. The size of individual dye molecules is very small (˜1 nm), which causes non-specific labeling and high background signals as dyes diffuse away from their intended targets. Spectrally, organic dyes tend to have fairly wide absorption and emission spectra (FWHM˜50 nm), which can lead to spectral overlap and re-absorption when using multiple dye species simultaneously. In normal use, dye molecules are exposed to a variety of harsh environments and often suffer from photobleaching and quenching due to the interactions with solvent molecules and reactive species such as oxygen or ions dissolved in solution.
In order to create more robust emitters with enhanced brightness and stability, researchers have developed composite nano- and micro-particles consisting of dye molecules and silica or polymer matrix. Thus the encapsulated dye molecules can be protected from external perturbations, with reducing stochastic blinking, photobleaching, and quenching. Dye-loaded polymer particles are superior to their silica counterparts in terms of the versatile chemical compositions, tunable surface chemistry suited for biocompatibility and bioconjugation, facile preparation, and easy control of the particle size and size distribution.
Gao et al. have incorporated pyrene dyes into polystyrene particles using a normal microemulsion approach, leading to a 40-fold increase in emission intensity with respect to the pure dye at the identical concentration (H. Gao et al., Colloid Polym. Sci. 2002, 280, 653). Dinsmore et al. swelled poly(methyl methacrylate) particles and absorbed a rhodamine dye into them for usage in a confocal microscopic study of colloidal dispersions (A. D. Dinsmore et al., Appl. Opt. 2001, 40, 4152). U.S. Pat. No. 5,716,855 disclosed fluorescent particles containing anthracene- or naphthacene-derivatived dyes aiming to the application as biological markers.
Up to now, most of the organic dyes commercially available, including the above mentioned dyes as well as ethidium bromide (U.S. Pat. No. 4,729,947, U.S. Pat. No. 5,346,603, U.S. Pat. No. 6,143,151, and U.S. Pat. No. 6,143,153), Nile red (U.S. Pat. No. 6,897,297 and U.S. Pat. No. 6,465,208), fluorescamine (U.S. Pat. No. 4,203,967), o-phthaldialdehyde (U.S. Pat. No. 6,969,615 and U.S. Pat. No. 6,607,918), cyanine dyes (U.S. Pat. No. 5,627,027 and U.S. Pat. No. 5,410,030), etc. are emissive only in their solution state, whereas emission is quenched in aggregation states (e.g., high dye concentration state, film state, solid state, etc.). This is attributed to the mechanism of nonradiative energy transfer between the closely packed chromophores, thus resulting in self-quenching of the fluorescence. Thus, the loading concentration of dyes in the polymer particles cannot be sufficiently high and accordingly the intensity of fluorescence is considerably limited.
With respect to polymers for dye encapsulation, the currently available species are mainly hydrophobic polystyrene and less hydrophobic poly(methyl methacrylate), as mentioned hereinabove. The hydrophobic nature of these particles commonly leads to clustering and non-specific binding of biological materials, which considerably limits their application in the aqueous environment of biology and other fields. Additionally, these particles are prepared and dispersed in organic solvent. For example, Hu et al. prepared poly(methyl methacrylate) fluorescent particles through dispersion polymerization in the mixture of hexane and ethanol (H. Hu et al., Langmuir 2004, 20, 7436). The solvent-dispersible polymer particles are difficult to disperse stably in aqueous media.
Recent studies on biomacromolecules aid the understanding of pathogenesis of numerous diseases as well as development of effective therapeutic agents. For example, fibrillation of amyloid proteins is recognized as a pathological hallmark of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Prion disease, and Huntington's disease. Insulin is a well-established model of amyloid fibrillation and its amyloid fibrils are found at frequent injection sites of diabetic patients and have been suggested as indicative of Parkinson's disease. Therefore, monitoring insulin aggregation and/or other amyloid proteins facilitates the understanding of pathogenesis of many neurodegenerative diseases or other diseases associated therewith, thereby developing effective diagnostic tools and therapeutic agents.
Amyloid fibril formation of insulin has been studied by a variety of spectroscopy and microscopy techniques including transmission electron microscopy (TEM), atomic force microscopy (AFM), real-time light scattering, stopped-flow turbidimetry, X-ray diffraction, fluorescence, circular dichroism (CD), and NMR spectroscopy. Among them, fluorescence technique is the most commonly used method on intrinsic fluorescence of proteins. For example, Thioflavin T (ThT) is a standard fluorescence probe for amyloid assay. Despite its widespread use, it suffers from a number of drawbacks, such as small stokes shift, low specificity, poor sensitivity, false-positive response, poor reliability, incapability of catching oligomeric intermediates, and unsuitability for kinetic study. Many of the other fluorophores for amyloid detection also contain electron donors and acceptors, between which intramolecular charge transfer occurs. Such fluorophores are sensitive to the hydrophobicity of the environment and their emissions are intensified upon binding to hydrophobic regions of amyloids rich in β-sheet structure. However, when multiple fluorophore molecules are accumulated in a hydrophobic patch of protein, π-π interaction between their stacked aromatic rings occurs, which promotes the formation of such detrimental species as excimers and exciplexes. This can lead to severe emission self-quenching, making the fluorophores unsuitable for quantitative analysis.
In addition to the problems in monitoring, insulin fibril formation has been a nuisance in delivery and long-term storage for treatment of diabetes because insulin can form amyloid fibrils in vitro under certain destabilizing conditions, such as elevated temperature, low pH, increased ionic strength and exposure to hydrophobic surface. It is also generally believed that dissociation of the native associated states of insulin (i.e. dimers, tetramers and hexamers) into monomers is a prerequisite for fibril formation. The monomers undergo partial unfolding into intermediate states, in which they re-associate into stable and fibrous amyloid aggregates. These destabilizing conditions lead to an early maturity of amyloid fibrils which does not favor the long-term storage and delivery of insulin. Therefore, inhibitors for protein aggregation are also of great significance in developing effective therapeutic agents for diabetes or for diseases which can be treated by similar biomolecules.
Accordingly, there is a need in the art for new sensors useful for detecting/sensing a wide variety of biomacromolecules. Sensors based on detecting fluorescence of an analyte such as a biomacromolecule are highly sensitive, thereby lowering detection limits. Sensors that have the capability to quantitatively analyze kinetics of biomacromolecules are desired. Notably, fluorescent markers which enable the monitor of amyloid fibrillation and compounds that inhibit fibrillation are most desired.